Effects of Life-Long Supplementation of Potassium Nitrate on Male Mice Longevity and Organs Pathology
, Sandrija Capkauskiene 3, Giedrius Trakimas 4,5, Kazimieras Pukenas 3 and Pranas Viskelis 6Round 1
Reviewer 1 Report
Effects of Life-long Supplementation of Potassium Nitrate on 2 Male Mice Longevity and Organs Pathology
In general, the paper is well-written and has interesting results, however, the conclusion is not well supported by the results.
In the material and methods section, the authors should mention that they looked for signs of cancer, if they did. And in the results, say whether or not lesions of this type were found. In order to reach a conclusion.
Comments for author File: 
Comments.pdf
Author Response
Dear Sir/Madam, we kindly thank you for your prompt feedback. Please see the answers to point by point below:
Reviewer #1:
1] In general, the paper is well-written and has interesting results, however, the conclusion is not well supported by the results.
Answer:
We generously thank you for the overall positive assessment of our manuscript, and hope that the revised text and modified table address all of the raised issues, and the conclusion is now better supported by the results.
2] In the material and methods section, the authors should mention that they looked for signs of cancer; if they did. And in the results, say whether or not lesions of this type were found. In order to reach a conclusion.
Answer:
Thank you for this comment. We have modified both the ‘Materials and Methods’ (lines 102-103) and the ‘Results’ (lines 138-139) sections as suggested.
3] Materials and methods,
Why did the authors used 12-16 weeks of age mice?
Answer:
Compliments for another good point! In order to avoid interactions of supplementation with developmental and growth peculiarities of individuals we choose matured mice. Matured mice considered to be at least three months old, although they are sexually mature by 35 days, relatively rapid maturational growth continues for most biological processes and structures until about three months. We have clarified this in the methods (lines 76-77).
4] Table 2, the column “all groups” is not understood. What means “all groups”
Answer: We excluded “All groups” column and modified the table adding the last row with the quantification of organ pathologies as suggested by another Reviewer and added an explanation in the Table 2 legend.
5] Line 152, which enzyme?
Answer: We indicated “a scavenging enzyme” as suggested (line 155).
6] Line 172, Why do you suggest that ”nitric oxide, coming with nitrates from food sources may delay pathological changes within the body and prolong life with no chronic conditions” If you used nitrate not from food sources.
Answer: We have modified the sentence using: “…nitrate supplementation” (line 175).
Author Response File: Author Response.pdf
Reviewer 2 Report
This is an interesting topic of investigation but the manuscript has some issues that must be attended to before considering for publication:
Introduction Section
The authors did not include the Justification nor Hypothesis. Those are imperative in research work. For this reason alone, it is worth considering that the work requires major recommendations.
Results and Discussion Sections
Lines 129- 135 and Table 2. The results of this paragraph are confusing. The sequence of the Table 2 explanation needs to be restructured to enhance the data analysis and this could change the perception of those results. Because in agreement with your statistical analysis, the KNO3 treatments only DELAY the organ pathologies but do not increase the life span. Life span sounds like an expectation and you performed a measurement, so the results have a number and an accurate definition.
Comments for author File: Comments.pdf
Author Response
Reviewer #2:
1] This is an interesting topic of investigation but the manuscript has some issues that must be attended before considering for publication:
Answer: We kindly thank you for your observations and remarks. Please find the answers to the questions and suggestions below.
Introduction Section
2] The authors did not include the Justification nor Hypothesis. Those are imperative in research work. For this reason alone, it is worth considering that the work requires major recommendations.
Answer: This is a good point! We have tried to clear it pointing to the novelty of our study (modifying the line 64). In the study we used an exploratory approach (with no direction of the possible effects in mind because there were no previous studies, on effects of this particular supplement on longevity of mice, known to us); as we wrote in the introduction: “…we aimed to analyze the long-term effects of…”. however, the sign of the hypotheses was not clearly stated. Thus, we have modified the statistical analysis section indicating two-tailed tests (hypotheses) used (line 110).
Results and Discussion Sections
3] Lines 129- 135 and Table 2. The results of this paragraph are confusing. The sequence of the Table 2 explanation needs to be restructured to enhance the data analysis and this could change the perception of those results. Because in agreement with your statistical analysis, the KNO3 treatments only DELAY the organ pathologies but do not increase the life span. Life span sounds like an expectation and you performed a measurement, so the results have a number and an accurate definition.
Answer: Thank you for this valuable comment. We have modified the table 2 adding the quantification of organ pathologies (mean organ pathologies - highlighted in bold) as suggested. However, we also left the longevity data, as we felt that this is also informative result. The unidirectional (two-tailed) test showed quite low probability (p=0.096, non-significant at the commonly used alfa level) that there were no changes in the life span, one-tailed probability would be p=0.048; Taking in mind the design of the study this is “promising” result, even the large (positive) effect of the KNO3 supplementation on overall survival is unlikely. We absolutely agree with your statement which raises questions and stimulates the discussion "increase of lifespan vs. delayed aging". However, we would kindly ask you to let us leave the results as they are in order to justify/stimulate future research and the discussion.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Effects of Life-long Supplementation of Potassium Nitrate on Male Mice Longevity and Organs Pathology
SECOND REVISIÓN
Again, the authors did not include the Justification nor Hypothesis. Those are imperative in research work. For this reason alone, it is worth considering that the work requires major recommendations.
Answer: This is a good point! We have tried to clear it pointing to the novelty of our study (modifying the line 64). In the study we used an exploratory approach (with no direction of the possible effects in mind because there were no previous studies, on the effects of this particular supplement on the longevity of mice, known to us); as we wrote in the introduction: “…we aimed to analyze the long-term effects of…”. however, the sign of the hypotheses was not clearly stated. Thus, we have modified the statistical analysis section indicating two-tailed tests (hypotheses) used (line 110).
Reviewer comments
Lines 63-67. Note that in your own answer (the above paragraph) you wrote the Justification... NOW, PLEASE WRITE IT IN THE MANUSCRIPT.
Line 110. Two-tailed test is a statistical method, it does not establish a hypothesis. Please, revise the basis for hypothesis establishments for research works of sciences and do adjustments as corresponds to the manuscript.
Results and Discussion Sections
3] Lines 129- 135 and Table 2. The results of this paragraph are confusing. The sequence of the Table 2 explanation needs to be restructured to enhance the data analysis and this could change the perception of those results. Because in agreement with your statistical analysis, the KNO3 treatments only DELAY the organ pathologies but do not increase the life span. Life span sounds like an expectation and you performed a measurement, so the results have a number and an accurate definition.
Answer: Thank you for this valuable comment. We have modified the table 2 adding the quantification of organ pathologies (mean organ pathologies - highlighted in bold) as suggested. However, we also left the longevity data, as we felt that this is also informative result. The unidirectional (two-tailed) test showed quite low probability (p=0.096, non-significant at the commonly used alfa level) that there were no changes in the life span, one-tailed probability would be p=0.048; Taking in mind the design of the study this is “promising” result, even the large (positive) effect of the KNO3 supplementation on overall survival is unlikely. We absolutely agree with your statement which raises questions and stimulates the discussion "increase of lifespan vs. delayed aging". However, we would kindly ask you to let us leave the results as they are in order to justify/stimulate future research and the discussion.
Reviewer comment
Of course, yes! but you discuss it and add the comments to the manuscript, it'll be an interesting proposal if it is well justified.
Author Response
RESPONSES TO SECOND REVISIÓN
Honourable Reviewer, on behalf of our whole scientific team once again let me thank you for dedicating your time to improve the quality of this manuscript. Please find the answers along with suggested corrections (updated manuscript file attached). Text blocks added in this round are in magenta, previous round corrections remain in blue.
Q. #1
Again, the authors did not include the Justification nor Hypothesis. Those are imperative in research work. For this reason alone, it is worth considering that the work requires major recommendations.
A. #1
Requested changes were added at the end of Introduction section.
Q. #2
"Of course, yes! but you discuss it and add the comments to the manuscript, it'll be an interesting proposal if it is well justified."
A. #2
Discussion section was modified as requested.
